Bottom Line:
A part of this process is decidualisation, comprising morphological and biochemical changes that result in formation of maternal stroma-derived decidual cells.Expression of the PGE2 receptors, PTGER2 and PTGER4, was clearly detectable.An in vitro decidualisation model with canine uterine stromal cells was successfully established, allowing future, more detailed studies to be undertaken on the underlying molecular and endocrine mechanisms of canine decidualisation.

Background: The uterine response to the presence of embryos is poorly understood in the domestic dog (Canis familiaris). The intimate embryo-maternal cross-talk, which begins following the hatching of blastocysts and embryo attachment leads to strong structural and functional remodelling of the uterus. A part of this process is decidualisation, comprising morphological and biochemical changes that result in formation of maternal stroma-derived decidual cells. These are an integral part of the canine placenta materna, which together with the maternal vascular endothelium are the only cells of the canine endotheliochorial placenta able to resist trophoblast invasion. These cells are also the only ones within the canine placenta expressing the progesterone receptor (PGR). Understanding the decidualisation process thus appears essential for understanding canine reproductive physiology.

Methods: Here, we investigated the capability of canine uterine stromal cells to decidualise in vitro, thereby serving as a canine model of decidualisation. A dbcAMP-mediated approach was chosen during a time course of 24 - 72 h. Tissue material from six (n = 6) healthy, dioestric bitches was used (approximately 2 weeks after ovulation). Cells were characterized by differential staining, nearly 100 % of which were vimentin-positive. Scanning and transmission electron microscope analyses were applied, and morphological changes were recorded with a live cell imaging microscope. Expression of several decidualisation markers was investigated.

Results: The in vitro cultured stromal cells acquired characteristics of decidual cells when incubated with 0.5 mM dbcAMP for 72 h. Their shape changed from elongated to rounded, while ultrastructural analysis revealed higher numbers of mitochondria and secretory follicles, and an increased proliferation rate. Elevated expression levels of IGF1, IGF2, PRLR and ERα were observed in decidualised cells; PRL and ERβ remained mostly below the detection limit, while PGR remained unaffected. The expression of smooth muscle α actin (αSMA), another decidualisation marker, was strongly induced. Among prostaglandin system members, levels of COX2 (PTGS2) and of PGE2-synthase (PTGES) were upregulated. Expression of the PGE2 receptors, PTGER2 and PTGER4, was clearly detectable.

Conclusion: An in vitro decidualisation model with canine uterine stromal cells was successfully established, allowing future, more detailed studies to be undertaken on the underlying molecular and endocrine mechanisms of canine decidualisation.

Fig8: Expression of cyclooxygenase 2 (COX2, PTGS2) and PGE2-synthase (PGES, PTGES) as determined by Western blot analysis. Following stimulation, cells were collected and homogenized and 20 μg of the lysate was used for Western blots. The averaged standardized optical density (SOD) is shown. GAPDH was used for loading control. The cell culture experiments were performed independently at least three times using cells isolated from different animals. Representative Western blots are shown. Student’s t-test was applied to test the effect of treatment on protein expression: in a (*) indicates P < 0.01, (**) indicates P < 0.004, (***) indicates P < 0.0001; in b (*) indicates P < 0.008

Mentions:
Similarly, as for EP4, the expression of PGT (SLCO2A1) remained unaffected (P > 0.05) during decidualisation. At the protein level, PTGS2 and PTGES expression profiles matched their respective mRNA levels, as shown in Fig. 8 for 24 h and 72 h of stimulation. Thus, whereas COX2 (PTGS2) expression already increased significantly (P < 0.01) at 24 h, both PTGS2 and PTGES were strongly upregulated at 72 h (P < 0.004 and p < 0.007, respectively), compared with their respective controls. Although the canine-specific anti-PGFS/AKR1C3 antibody previously tested positively on canine uteri and placenta [33], within the current study the expression of PGFS/AKR1C3 protein remained below the detection limit (not shown).Fig. 8

Fig8: Expression of cyclooxygenase 2 (COX2, PTGS2) and PGE2-synthase (PGES, PTGES) as determined by Western blot analysis. Following stimulation, cells were collected and homogenized and 20 μg of the lysate was used for Western blots. The averaged standardized optical density (SOD) is shown. GAPDH was used for loading control. The cell culture experiments were performed independently at least three times using cells isolated from different animals. Representative Western blots are shown. Student’s t-test was applied to test the effect of treatment on protein expression: in a (*) indicates P < 0.01, (**) indicates P < 0.004, (***) indicates P < 0.0001; in b (*) indicates P < 0.008

Mentions:
Similarly, as for EP4, the expression of PGT (SLCO2A1) remained unaffected (P > 0.05) during decidualisation. At the protein level, PTGS2 and PTGES expression profiles matched their respective mRNA levels, as shown in Fig. 8 for 24 h and 72 h of stimulation. Thus, whereas COX2 (PTGS2) expression already increased significantly (P < 0.01) at 24 h, both PTGS2 and PTGES were strongly upregulated at 72 h (P < 0.004 and p < 0.007, respectively), compared with their respective controls. Although the canine-specific anti-PGFS/AKR1C3 antibody previously tested positively on canine uteri and placenta [33], within the current study the expression of PGFS/AKR1C3 protein remained below the detection limit (not shown).Fig. 8

Bottom Line:
A part of this process is decidualisation, comprising morphological and biochemical changes that result in formation of maternal stroma-derived decidual cells.Expression of the PGE2 receptors, PTGER2 and PTGER4, was clearly detectable.An in vitro decidualisation model with canine uterine stromal cells was successfully established, allowing future, more detailed studies to be undertaken on the underlying molecular and endocrine mechanisms of canine decidualisation.

Background: The uterine response to the presence of embryos is poorly understood in the domestic dog (Canis familiaris). The intimate embryo-maternal cross-talk, which begins following the hatching of blastocysts and embryo attachment leads to strong structural and functional remodelling of the uterus. A part of this process is decidualisation, comprising morphological and biochemical changes that result in formation of maternal stroma-derived decidual cells. These are an integral part of the canine placenta materna, which together with the maternal vascular endothelium are the only cells of the canine endotheliochorial placenta able to resist trophoblast invasion. These cells are also the only ones within the canine placenta expressing the progesterone receptor (PGR). Understanding the decidualisation process thus appears essential for understanding canine reproductive physiology.

Methods: Here, we investigated the capability of canine uterine stromal cells to decidualise in vitro, thereby serving as a canine model of decidualisation. A dbcAMP-mediated approach was chosen during a time course of 24 - 72 h. Tissue material from six (n = 6) healthy, dioestric bitches was used (approximately 2 weeks after ovulation). Cells were characterized by differential staining, nearly 100 % of which were vimentin-positive. Scanning and transmission electron microscope analyses were applied, and morphological changes were recorded with a live cell imaging microscope. Expression of several decidualisation markers was investigated.

Results: The in vitro cultured stromal cells acquired characteristics of decidual cells when incubated with 0.5 mM dbcAMP for 72 h. Their shape changed from elongated to rounded, while ultrastructural analysis revealed higher numbers of mitochondria and secretory follicles, and an increased proliferation rate. Elevated expression levels of IGF1, IGF2, PRLR and ERα were observed in decidualised cells; PRL and ERβ remained mostly below the detection limit, while PGR remained unaffected. The expression of smooth muscle α actin (αSMA), another decidualisation marker, was strongly induced. Among prostaglandin system members, levels of COX2 (PTGS2) and of PGE2-synthase (PTGES) were upregulated. Expression of the PGE2 receptors, PTGER2 and PTGER4, was clearly detectable.

Conclusion: An in vitro decidualisation model with canine uterine stromal cells was successfully established, allowing future, more detailed studies to be undertaken on the underlying molecular and endocrine mechanisms of canine decidualisation.